(Supported by NIH NIAID U01 AI43020 to J. Keithly) This is a collaborative effort with the Wadsworth EM Core Facility and the BMIRR to determine by HVEM and 3D tomography whether a mitochondrion, plastid and/or other organelle is present in the protozoon Cryptosporidium parvum. If a unique plant-like organelle can be shown to carry out metabolic functions essential for the parasite, then chemotherapy specific for the parasite and not its human host would be possible. Currently, there is no treatment for cryptosporidiosis, a potentially fatal diarrheal illness of the immuno-compromised (including children, the elderly, and cancer and HIV-positive patients). Preliminary ultrastructural studies performed by Susan Langreth indicated that in the posterior of excysted sporozoites, there are several unusual organelles: one for storage, another resembling the phycobiliproteins or stacked thylakoids of algae, and perhaps a mitochondrial remnant. We hope to determine what these organelles are. To provide additional evidence of the function of these organelles, we hope to confirm the location and presence of two proteins previously localized by indirect fluorescent antibody labeling in sporozoites of C. parvum: a Ca2+ P-ATPase localized in both apical and perinuclear regions of the parasite (Mol. Biochem. Parasitol. 90:307-16, 1997) and a fatty acid synthetase encoded by the nucleus but targeted to the apicoplast (primary chloroplast) in Plasmodium and Toxoplasma. In conjunction with these ultrastructural studies, the Wadsworth Biochemistry Core Facility, will provide analysis of sporozoites for the presence of eukaryotic and prokaryotic pigments e.g., chlorophyll, phycoerythyrin, and phycocyanin. Two double-tilt series tomographic reconstructions were made of 0.25 (m plastic sections, one recorded on the HVEM and one on the IVEM. The organelle in the posterior end of the protozoon has still not been positively identified. Several images were prepared for submission with a grant proposal supplement. There is also interest in studying membranes found in the anterior end of the cell. These membranes have been observed in similar cells such as Toxoplasma but have not been described in Crytosporidium. Dr. Keithly felt that serial thin-section reconstruction of the membranes using Sterecon would be preferable to tomography because of the complexity of the membranes, as well as the identification difficulties of these membranes in thicker sections.